1. Volume 19, Issue 4, Pages 523-534 (August 2005)
The Bromodomain Protein Brd4 Is a Positive Regulatory Component of P-TEFb and Stimulates RNA Polymerase II-Dependent Transcription 
Moon Kyoo Jang, Kazuki Mochizuki, Meisheng Zhou, Ho-Sang Jeong, John N. Brady, Keiko Ozato 
Molecular Cell 
Volume 19, Issue 4, Pages (August 2005)
DOI: /j.molcel
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2. Figure 1 Brd4 Interacts with CyclinT1/Cdk9
(A) Two-step purification scheme to identify Brd4-associated proteins.
(B) Immunopurified h-Brd4-f complexes were fractionated on SDS-PAGE. Bands were analyzed by mass spectrometry and immunoblotting. Lanes 1 and 2, colloidal-blue staining. The positions of h-Brd4-f, P-TEFb subunits, and components of TRAP complex are marked by filled arrows and open arrows, respectively. Molecular size markers are shown on the right. Lanes 3–8, the complexes were immunoblotted with indicated antibodies.
(C) HeLaS3 NEs without dialysis were tested for coprecipitation of Brd4 and P-TEFb as in (B).
(D) TCEs with h-Brd4-f or control (FLAG-HA alone) were tested for coprecipitation. The precipitates were analyzed by immunoblotting with antibodies shown on the left. Abbreviations: TCE, input for total cell extracts and IP, FLAG immunoprecipitates.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2005 Elsevier Inc. Terms and Conditions

3. Figure 2 Endogenous Brd4 Interacts with CyclinT1 and Cdk9
(A) HeLaS3 NEs and immunoprecipitates purified as in Figure 1A were analyzed by immunoblotting with additional antibodies.
(B) NEs from HeLa cells transiently transfected with GFP constructs were precipitated by anti-GFP antibody, and input NEs (left, NE) or precipitates (right, IP) were analyzed by immunoblotting with antibodies shown on the left.
(C) HeLa NEs were precipitated with preimmune or anti-Brd4 antibody, shown on the top (IP), and precipitates were immunoblotted with antibodies, shown on the left.
(D) HeLa NEs were precipitated with the antibodies, shown at the top (IP), and precipitates were immunoblotted with antibodies, shown on the left.
(E) HeLa NEs were passed through preimmune or anti-Brd4 antibody beads three times, and supernatants were immunoblotted with antibodies, shown on the left.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3 Domain Requirement
(A) Diagram of cyclinT1 deletions. Brd4 binding data are summarized on the right.
(B) GST-fusions were incubated with radiolabeled wt or ΔC-term Brd4, and bound materials were detected by autoradiography (top). Coomassie blue staining of GST-cyclinT1 proteins is shown at the bottom.
(C) Diagram of Brd4 deletions. CyclinT1 binding data are summarized on the right.
(D) Wt and Brd4 deletions tagged with 6xHis and FLAG and purified using baculoviral system were incubated with GST or GST-cyclinT1 beads, and bound materials were immunoblotted with anti-FLAG antibody (top). Coomassie blue staining of GST proteins is shown at the bottom.
(E) HeLa NEs containing FLAG, FLAG-tagged Brd4, or deletions were precipitated with anti-FLAG antibody and immunoblotted with indicated antibodies at left.
(F) HeLa NEs with FLAG alone, FLAG-tagged wt cyclinT1, or Δ(426–516) were precipitated by anti-FLAG antibody and immunoblotted with indicated antibodies at the left.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4 Interaction of Brd4 and CyclinT1 in Living Cells
(A) NIH3T3 cells transfected with GFP-cyclinT1 were stained with antibodies for Brd4 and phospho-RNAPII (Ser2) and viewed on a confocal microscope. All three proteins localized to nonnucleolar space in the nucleus with varied speckle-like structures. The triple merge image indicates patchy colocalization of the three proteins.
(B) HeLa cells were transfected with indicated combinations of YN and YC constructs along with pEBFP. BiFC signals were viewed as above. Blue images on upper panels indicate BFP run as transfection control. Intense YFP signals in the nucleus represent BiFC.
(C) Expression of Brd4-YC and YN-cyclinT1 was detected by immunoprecipitation and immunoblot with anti-GFP antibody.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5 Brd4 Interacts with 7SK snRNA-Free CyclinT1/Cdk9
(A) NEs (0.5 mg) from HeLaS3 cells expressing h-Brd4-f were separated on a glycerol gradient centrifugation into 21 fractions and were analyzed by immunoblot or RT-PCR for indicated proteins/RNA, shown on the left. Estimated molecular weights of protein complexes were shown on the right.
(B) HeLa NEs containing h-Brd4-f were precipitated with antibodies, shown at the top, and analyzed by semiquantitative RT-PCR, real-time RT-PCR (bottom) and immunoblot, with antibodies shown on the left.
(C) Extracts from parental NIH3T3 or cells expressing control or Brd4-siRNA were precipitated with anti-Cdk9 antibody and analyzed as in (A) except by using primers for mouse 7SK snRNA.
(D) CyclinT1-Cdk9 complexes precipitated from HeLa NEs (400 μl) were incubated with ∼1 μg of purified recombinant wt or mutant Brd4 or BSA (as control) for 1 hr at 4°C. 7SK snRNA in supernatants was detected by semiquantitative and real-time RT-PCR.
Results indicate the average of three independent experiments ± SD in (B)–(D).
(E) NIH3T3 NEs (0.3 mg) with control or Brd4-siRNA were analyzed by glycerol gradient centrifugation as in (A), and immunoblot for indicated proteins, shown on the left.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 6
Brd4 Stimulates RNAPII CTD Phosphorylation and Enhances Promoter Activity
(A) Total extracts from HeLa cells transfected with indicated vectors (top) were immunoblotted with antibodies specific for phospho-Ser2 (H5) or phospho-Ser5 (H14) of RNAPII CTD. Immunoblots with additional antibodies were run as controls for transfection and loading.
(B) NIH3T3 cells expressing Brd4-siRNA or control-siRNA were transfected with increasing amounts of Brd4 vector, and total extracts were immunoblotted as in (A).
(C) HeLa cells were cotransfected with 20 ng of indicated luciferase reporter and 0.1 μg or 0.4 μg of pFLAG-CMV2-Brd4 vector for 24 hr. In all reporter assays, the amount of transfected DNA was kept constant by adding appropriate empty vectors. Shown at the bottom are immunoblot detection of Brd4, cyclinT1, Cdk9, and α-tubulin as controls for transfection and loading.
(D) HeLa cells were cotransfected with 20 ng of HIV-1 LTR reporter, 0.1 μg of pFLAG-CMV2-Brd4, and 0.4 μg of pcDNA3-cyclinT1 vectors. Luciferase assay and immunoblots were performed as in (C).
(E) HeLa cells were transfected with the HIV-1 LTR reporter and 0.1 or 0.25 ng of wt Brd4 or deletion vectors.
(F) Parental NIH3T3 cells or cells expressing control-siRNA or Brd4-siRNA were transfected with the HIV-1 LTR reporter and 0.5 μg of Brd4 vector.
Luciferase activities are expressed as relative units obtained from the average of three assays ± SD in (C)–(F).
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 7
Brd4 Enhances the Recruitment of P-TEFb to the Integrated HIV-1 LTR Reporter
(A) Diagram of the HIV-1 LTR reporter stably integrated into NIH3T3 cells and the position of the primers tested for ChIP analysis.
(B) NIH3T3 cells with the integrated HIV-1 LTR reporter were transduced with control or Brd4-siRNA and selected by puromycin (2 μg/ml) for 4 days. HIV-1 LTR reporter activity and Brd4 expression were tested by luciferase assay and immunoblotting. Luciferase activities are expressed as relative units obtained from the average of three assays ± SD.
(C) Brd4 and Cdk9 bind to the integrated HIV-LTR sequence in vivo. NIH3T3 with control or Brd4-siRNA were subjected to ChIP assay by using anti-Cdk9 and anti-Brd4 antibodies shown on the top. Results indicate the average of two independent assays ± SD.
(D) NIH3T3 cells with the integrated HIV-1 LTR reporter were transduced as shown in (B). After treatment with TSA for 6 hr, HIV-1 LTR reporter activity was tested by luciferase assay. Luciferase activities are expressed as relative units obtained from the average of three assays ± SD.
(E) P-TEFb binding is regulated by Brd4 and acetylation of chromatin. NIH3T3 cells with control or Brd4-siRNA were treated with TSA (200 ng/ml) for 4 hr and subjected to ChIP assay by using anti-Cdk9, anti-Brd4, and anti-tetra acetyl-histone H4 antibodies shown on the top. Real-time RT-PCR was performed with a primer set (LTR-Luc139). Results indicate the average of two independent assays ± SD.
(F) A model for Brd4-P-TEFb interaction. Core P-TEFb (cyclinT1 and Cdk9) occurs in association with either Brd4 or the inhibitory subunit, representing the positive or negative regulatory component, respectively. P-TEFb activity is maintained at equilibrium by reversible exchange of the two subunits. By binding to acetylated chromatin, Brd4 facilitates the recruitment of P-TEFb to promoters and enhances their transcription. Interaction of Brd4 with the Mediator complex also increases P-TEFb recruitment.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2005 Elsevier Inc. Terms and Conditions